PROJECT SUMMARY This Predoctoral award proposal is submitted to support the long-term goal of unraveling the mechanisms by which specific subsets of neurons die in neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), an incurable adult-onset fatal paralytic disorder. This will be achieved by studying the involvement of non-neuronal cells in the death of motor neurons (MNs) in ALS which is now well-recognized. Relevant to this goal, the Przedborski lab has found that astrocytes prepared from a mutant superoxide dismutase 1 (mSOD1) transgenic mouse model of ALS kill cultured MNs through a toxic event whose nature remains to be defined. Subsequent investigations support the notion that ALS astrocyte toxicity might result from the aberrant trafficking of the amyloid precursor protein (APP) due to a retromer defect. Although dysfunction of the retromer, leading to an aberrant processing of APP, has been proposed in several neurodegenerative disorders, whether and how retromer dysfunction in astrocytes may contribute to MN degeneration in ALS is not known. Therefore, in this project, which seeks to elucidate the role of retromer in ALS, we propose three Aims. In Aim 1, to gain insights into the mechanism by which retromer deficiency results in non-cell autonomous MN death, we will characterize the effect of retromer trafficking dysfunction in astrocytes in vitro by using RNA interference against Vps35?a core component of the retromer?and examining changes in i) APP fragment levels through immunoblotting, ii) APP trafficking through confocal and live imaging, and iii) MN toxicity through immunocytochemistry. In Aim 2, we will assess the behavioral and neuropathological alterations in the mouse brain, with specific attention to the spinal MN pathway, following an astrocyte-specific retromer defect. For this experiment, Vps35Fl/Fl mutant mice will be crossed with transgenic Aldh1l1-Cre mice to provide an astrocyte-specific deletion of Vps35. Finally, in Aim 3, we will use the small molecule R33, which stabilizes the retromer complex, to determine whether such a preclinical therapeutic strategy can modify the ALS-like phenotype seen in transgenic mSOD1 mice. This project focuses on the role of the retromer in astrocytes, which has not been done before, uses a unique mouse line and small molecule, and studies ALS. Should the project be successful, our results may have far-reaching implications for both our understanding and treatment of this dreadful neurodegenerative disorder.